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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

I present an electron microscopical analysis of the columnar hexagonal liquid crystalline phase of DNA. Freeze-fracture methods reveal that this phase is a lamellar structure, each layer (30 to 40 A thick) composed of DNA molecules aligned in parallel. Numerous defects can be seen in the structure, and their nature is determined. I show that they are mainly screw dislocations of both handedness. By this method it is possible to follow individual double-stranded DNA molecules in this highly packed structure. I show, moreover, that there is a local twist between DNA molecules along the screw dislocation lines and that this twist can be either right-handed or left-handed. The interest of such ultrastructural analysis is discussed in relation to the understanding of chromatin structure.
J Mol Biol 1991 Mar 05
PMID:Supramolecular organization of double-stranded DNA molecules in the columnar hexagonal liquid crystalline phase. An electron microscopic analysis using freeze-fracture methods. 200

The substrate specificity of thermophilic xylose isomerase from Clostridium thermosulfurogenes was examined by using predictions from the known crystal structure of the Arthrobacter enzyme and site-directed mutagenesis of the thermophile xylA gene. The orientation of glucose as a substrate in the active site of the thermophilic enzyme was modeled to position the C-6 end of hexose toward His-101 in the substrate-binding pocket. The locations of Met-87, Thr-89, Val-134, and Glu-180, which contact the C-6-OH group of the substrate in the sorbitol-bound xylose isomerase from Arthrobacter [Collyer, C.A., Henrick, K. & Blow, D. M. (1990) J. Mol. Biol. 212, 211-235], are equivalent to those of Trp-139, Thr-141, Val-186, and Glu-232 in the thermophilic enzyme. Replacement of Trp-139 with Phe reduced the Km and enhanced the kcat of the mutant thermophilic enzyme toward glucose, whereas this substitution reversed the effect toward xylose. Replacement of Val-186 with Thr also enhanced the catalytic efficiency of the enzyme toward glucose. Double mutants with replacements Trp-139----Phe/Val-186----Thr and Trp-139----Phe/Val-186----Ser had a higher catalytic efficiency (kcat/Km) for glucose than the wild-type enzyme of 5- and 2-fold, respectively. They also exhibited 1.5- and 3-fold higher catalytic efficiency for D-glucose than for D-xylose, respectively. These results provide evidence that alteration in substrate specificity of factitious thermophilic xylose isomerases can be achieved by designing reduced steric constraints and enhanced hydrogen-bonding capacity for glucose in the substrate-binding pocket of the active site.
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PMID:Switching substrate preference of thermophilic xylose isomerase from D-xylose to D-glucose by redesigning the substrate binding pocket. 202 50

Freeze-fracture electron microscopy reveals that intramembrane particles are concentrated in a band encircling the posterior portion of the acrosome of Strongylocentrotus purpuratus sperm. Two colloidal gold labeling methods, label-fracture and replica-staining fracture-flip, were employed to show that the plant lectin wheat germ agglutinin, which recognizes a 210 kDa sperm surface glycoprotein, binds to this localized band of intramembrane particles. Monoclonal antibody J18/2, which also recognizes the 210 kDa surface glycoprotein, shows this localized binding in approximately 20% of the sperm observed in this study. The majority of sperm displayed a uniform distribution of receptor sites for monoclonal antibody J18/2. Since wheat germ agglutinin and monoclonal antibody J18/2 are known to agglutinate Strongylocentrotus purpuratus sperm but not sperm of another sea urchin, Lytechinus pictus, similar determinations were made for the latter species. Lytechinus pictus sperm are not labeled with wheat germ agglutinin and are only sparsely labeled with monoclonal antibody J18/2. The acrosomal localizations of wheat germ agglutinin and monoclonal antibody J18/2 receptors in Strongylocentrotus purpuratus sperm are consistent with the involvement of the 210 kDa surface glycoprotein in an egg jelly-induced sperm acrosome reaction. Low-temperature post-embed labeling of thin sections with wheat germ agglutinin and monoclonal antibody J18/2 show concentrations of label within the acrosomal vesicle of Strongylocentrotus purpuratus sperm, suggesting the presence of an intracellular storage site for the 210 kDa glycoprotein.
Mol Reprod Dev 1991 Apr
PMID:Localization of wheat germ agglutinin and antibody binding sites on the plasma membranes of sea urchin sperm heads as revealed by label-fracture and fracture-flip. 206 84

To study the effect of protein flexibility on electrostatic recognition, we have devised two novel computer graphic representations of the changes in the electrostatic field of a protein resulting from its internal motions. The atomic structure of Cu, Zn superoxide dismutase was minimized, and the 200 lowest frequency normal modes of the enzyme were determined. Individual and combined normal-mode vibrations were visualized interactively with the program Flex. Normal-mode motions are fast enough (approximately 10(-11) s cycle-1) to evade solvent damping, thus allowing long-range electrostatic interactions to dominate. The changing electrostatic environment of the protein was examined by animating precalculated frames of electrostatic field vectors with GRAMPS. With Vu, changes in electrostatic potential were displayed as variations in the color-coding of dots lying on a consensus surface that maintains the protein's shape. The consensus surface was calculated with the program Sphinx, and was derived from spherical harmonic approximations of expanded molecular surfaces. The ability to view the effects of molecular motions interactively should be useful in understanding the relationships of protein structure to function.
J Mol Graph 1990 Sep
PMID:Visualization of molecular flexibility and its effects on electrostatic recognition. 227 8

Freeze-etch electron microscopy of pure RecA protein aggregates, as well as of RecA protein complexes on single-stranded and double-stranded DNA formed with various nucleotides, has permitted a clearer discrimination between the two different helical polymers that this protein forms. Both are continuous, single-start, right-handed helices; however, the form observed when ATP or non-hydrolyzable ATP analogs are present has a pitch of 9.5 nm and a diameter of 10 nm, while the other form, observed in the absence of ATP or its analogs, or in the presence of ADP, has a pitch of 6 nm and a diameter of 12 nm. The former "long pitch" helix is found only when RecA protein is bound to DNA. The latter "short pitch" helix is also observed in pure RecA protein polymers (also termed rods) and in the needle-like paracrystals of RecA protein that form in the presence of magnesium or spermidine ions, representing bundles of rods closely packed in register. Addition of ATP or non-hydrolyzable ATP analogs in the absence of DNA dissociates the pure RecA protein crystals, as well as individual helical rods, into short curvilinear chains of attached monomers. These chains typically form closed, circular rings of 7(+/- 1) protein monomers, similar in construction to a single turn of the RecA protein helix, but significantly broader in diameter. The role of ATP in interconverting the various polymeric forms of RecA protein is discussed within the context that ATP functions as a reversible allosteric effector of RecA protein, much as it mediates reversible conformational changes in other vectoral motor proteins such as myosin, dynein, kinesin and the 70,000 Mr "heat shock" ATPases. We discuss how cyclic conversions back and forth between the short- and long-pitch conformations of RecA protein could mediate in reversible single-stranded and double-stranded DNA interactions during the search for homology.
J Mol Biol 1989 Dec 05
PMID:Visualization of RecA protein and its complexes with DNA by quick-freeze/deep-etch electron microscopy. 269 35

Pullulanase, a secreted lipoprotein of Klebsiella pneumoniae, is initially localized to the outer face of the outer membrane, as shown by protease and substrate accessibility and by immunofluorescence tests. Freeze-thaw disruption of these cells released both membrane-associated and apparently soluble forms of pullulanase. Membrane-associated pullulanase co-fractionated with authentic outer membrane vesicles upon isopycnic sucrose-gradient centrifugation, whereas the quasi-soluble form had the same equilibrium density as inner membrane vesicles and extracellular pullulanase aggregates. The latter also contained outer membrane maltoporin, but were largely devoid of other membrane components including LPS and lipids. K. pneumoniae carrying multiple copies of the pullulanase structural gene (pulA) produced increased amounts of cell-associated and secreted pullulanase, but a large proportion of the enzyme was neither exposed on the cell surface nor released into the medium, even after prolonged incubation. This suggests that factors necessary for pullulanase secretion were saturated by the over-produced pullulanase. When pulA was expressed under lacZ promotor control, the pullulanase which was produced was not exposed on the cell surface at any time, suggesting that pullulanase secretion genes are not expressed constitutively, and raising the possibility that they, like pulA, may be part of the maltose regulon.
Mol Microbiol 1987 Jul
PMID:Export and secretion of the lipoprotein pullulanase by Klebsiella pneumoniae. 283 22

Activated ras oncogenes have previously been implicated in the pathogenesis of human lung carcinomas. A v-Ha-ras-containing retrovirus, Zip-ras, was generated by inserting the coding region of the v-Ha-ras oncogene into the Zip-NeoSV(X) [Cepko et al., Cell 37:1053-1062, 1984] retroviral vector. Amphotrophic Zip-ras retrovirus was used to infect an SV40 large T antigen-positive immortalized cell line, BEAS-2B, derived from normal bronchial epithelial cells, the predominant progenitor cells of human lung carcinomas. Zip-ras-infected BEAS-2B cells selected for G418 resistance formed anaplastic carcinomas in 12 of 15 athymic nude mice (latency 3 wk), whereas Zip-NeoSV(X)-infected BEAS-2B control cultures inoculated into 12 nude mice formed no tumors after a minimum of 7 mo. Tumor cell lines were established and demonstrated to be of human epithelial origin and to express v-Ha-ras p21 protein. A common feature of the tumor cell lines was an increase in ploidy. The increased efficiency of neoplastic transformation by v-Ha-ras of cell lines as compared with our previous results with normal bronchial epithelial cells [Yoakum et al., Science 227:1174-1179, 1985] is consistent with the hypothesis that the "immortalization" step is rate-limiting in in vitro human epithelial cell carcinogenesis.
Mol Carcinog 1988
PMID:Neoplastic transformation of a human bronchial epithelial cell line by a recombinant retrovirus encoding viral Harvey ras. 285 21

In the present paper rat hepatocytes in primary monolayer culture were used to investigate the adverse effects of chlorpromazine (CPZ) at the cellular level. As revealed by thin sectioning many of the ultrastructural alterations were comparable to those described for the isolated perfused rat liver under the influence of CPZ. Alterations comprised short-term effects, such as dilation of the rough endoplasmic reticulum and the nuclear envelope, and long-term effects including huge accumulations of myeloid bodies within the cytoplasm as well as dilation and diverticulation of bile canaliculi. Freeze-fracturing revealed the dislocation of intramembrane particles in the sinusoidal plasma membrane which could be detected as early as 30 min after exposure to CPZ. As judged from filipin cytochemistry, alterations in the cholesterol content seems to play a minor role in the process of membrane damage except at the sinusoidal surface where a reduction of cholesterol content may contribute to the impairment of membrane functions. It is concluded that CPZ exerts its cholestatic effect primarily by a rapid disturbance of the membrane architecture of the sinusoidal surface and secondarily by other interactions with the bile secretory apparatus.
Virchows Arch B Cell Pathol Incl Mol Pathol 1985
PMID:Primary culture of rat hepatocytes as a model system of canalicular development, biliary secretion, and intrahepatic cholestasis. V. Disturbance of the cellular membrane and bile canalicular ultrastructure induced by chlorpromazine. 286 34

The ultrastructure of gap junctions between rat liver parenchymal cells has been studied after in vivo ischemia, with and without subsequent blood reflow. Freeze fracture replicas were analysed by electron microscopic observation, optical diffraction and morphometric analysis. In control specimens gap junction connexons were widely dispersed and arranged in nearly random fashion over nearly the whole junctional area, with only minute spots of hexagonal connexon arrangement. An ischemic period of 30 min, from which the vast majority of cells are capable of recovery after restoration of the blood supply, usually entails only a slight enlargement of the areas of hexagonally arranged connexons. After 120 min of ischemia without reflow, which results in necrosis of most parenchymal cells, all gap junctions showed a completely hexagonal arrangement of connexons. The numerical density of connexons after 30 and 120 min of ischemia without reflow was significantly higher than in controls, whereas after 30 min of ischemia followed by 2 h of reflow the numerical density had returned to control levels. A fully hexagonal arrangement of gap junction connexons, as occurs after longer periods of ischemia, seems to be related to irreversible cell damage and presumably to metabolic uncoupling of cells. This was preceded by an increase in the numerical density of connexons, which is probably a reversible phenomenon.
Virchows Arch B Cell Pathol Incl Mol Pathol 1987
PMID:Gap junction ultrastructure in rat liver parenchymal cells after in vivo ischemia. 289 Dec 18

We have measured by radioimmunoassay the amount of total, free, and bound forms of cyclic AMP (cAMP) within the abdominal ganglion and in five identified cell bodies of neurons from Aplysia californica. In the abdominal ganglion the unbound (free) cAMP levels comprised approximately 25-30% of the total cAMP content under the unstimulated condition, i.e., bathed in high-magnesium saline. Under pharmacological conditions that blocked endogenous phosphodiesterase and activated adenylate cyclase, ganglionic free cAMP levels were elevated more than fourfold, while bound cAMP levels more than doubled. Freeze-substitution techniques were employed to facilitate isolation of individual cell bodies either before or after pharmacological manipulation of cAMP levels. The basal, free cAMP content of cells R2, LP1, R15, L11, and L2-L6 was in the range of 10-40 pmol/mg of cell protein, which accounted for approximately one-half of the total cAMP content per cell body. Determinations of individual cell volumes indicated that the basal, free cAMP concentrations ranged from 1 to 6 microM. Under the same pharmacological conditions that elevated ganglionic cAMP in levels, no changes were measured in either the free or the bound forms of cAMP in isolated cell bodies. Our results indicate that the cAMP elevation was compartmentalized within the neuropilar region of the ganglion, most likely within the processes of the nerve cells. Previous results demonstrated that cAMP injections into the same Aplysia neurons studied here induced a cAMP-activated sodium current, INa (cAMP). In this report we discuss the possibility that pharmacological elevation of cAMP within neuronal processes may reach concentrations similar to those produced by cAMP injections into somata.
Cell Mol Neurobiol 1987 Mar
PMID:Compartmentalization of cyclic AMP elevation in neurons of Aplysia californica. 303 61


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